ALPK1 missense pathogenic variant in five families leads to ROSAH syndrome, an ocular multisystem autosomal dominant disorder

Abstract

Purpose: To identify the molecular cause in five unrelated families with a distinct autosomal dominant ocular systemic disorder we called ROSAH syndrome due to clinical features of retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache.

Methods: Independent discovery exome and genome sequencing in families 1, 2, and 3, and confirmation in families 4 and 5. Expression of wild-type messenger RNA and protein in human and mouse tissues and cell lines. Ciliary assays in fibroblasts from affected and unaffected family members.

Results: We found the heterozygous missense variant in the ɑ-kinase gene, ALPK1, (c.710C>T, [p.Thr237Met]), segregated with disease in all five families. All patients shared the ROSAH phenotype with additional low-grade ocular inflammation, pancytopenia, recurrent infections, and mild renal impairment in some. ALPK1 was notably expressed in retina, retinal pigment epithelium, and optic nerve, with immunofluorescence indicating localization to the basal body of the connecting cilium of the photoreceptors, and presence in the sweat glands. Immunocytofluorescence revealed expression at the centrioles and spindle poles during metaphase, and at the base of the primary cilium. Affected family member fibroblasts demonstrated defective ciliogenesis.

Conclusion: Heterozygosity for ALPK1, p.Thr237Met leads to ROSAH syndrome, an autosomal dominant ocular systemic disorder.

Keywords: ALPK1; ROSAH syndrome; ciliogenesis; genome sequencing; retinal dystrophy.

Fig. 1

Pedigree of families with ROSAH syndrome and pathogenic variant identification in ALPK1. (a) Five families affected by autosomal dominant ROSAH syndrome were analyzed in this study, from Utah (USA), Australia, The Netherlands, Virginia (USA), and Delaware (USA). Affected individuals are shaded and those examined by exome sequencing and genome sequencing in the Utah, Australia, and Netherlands families are indicated with an asterisk. Variant status is indicated below each individual for whom sequencing data was obtained (T237M indicates allele coding for [p.Thr237Met]; + indicates normal allele). (b) Subsequent Sanger sequencing in all families confirmed segregation of the ALPK1 (NM_025144) variant with disease. All individuals with the variant in ALPK1 have the ROSAH syndrome phenotype. The c.710C base pair (bp) is indicated by the arrow, and the bp 710–711 CG dimer is indicated by the thickened line. (c) Exonic structure of ALPK1 (NM_025144) with the pathogenic variant on exon 9 identified in ROSAH syndrome. (d) Protein structure of ɑ-kinase 1 showing the location of the pathogenic variant and the location of the known kinase region. (e) Comparison across species for ALPK1 Thr237 and flanking sequences using multiple sequence alignment from UniProtKB/UniRef100 Release 2011_12 (14 December 2011) database. A high degree of conservation of the Thr237 amino acid in vertebrates is evident. Flanking sequences are also highly conserved. Shaded areas represent consensus sequence. Red letters represent deviation from consensus.

Fig. 2

Clinical images for patients III.2 and II.3 from the Australian cohort and II.1 from the Delaware cohort.(a–d) Fundus imaging for patient III.2, Australian cohort. (a) Swollen optic disc with swelling extending onto the peripapillary nerve fiber layer. (b) Fundus image showing the vitreous and preretinal hemorrhage. (c) Ultrawide field fundus images color corrected with Optos viewing software (Optos plc, Dunfermline, Scotland, UK) shows prominence of the nerve fiber layer, early retinal atrophy particularly just anterior to the vascular arcades. No pigmentary changes are seen. (d) Ultrawide field fundus autofluoresence (FAF) (Optos plc, Dunfermline, Scotland, UK) showing diffuse hyperautofluoresence in a broad ring extending anterior to the vascular arcades and loss of the normal foveal hypoautofluorescence. (e, g) OCT. (e) Patient III.2 from Australian cohort shows retinal and peripapillary generalized thickening with indistinct retinal layers. The macula shows cystic changes and loss of the external limiting membrane (ELM) and photoreceptor inner segment/outer segment (IS-OS) junction. (g) Age-matched normal OCT is shown for comparison. (f, h) Visual electrophysiology. (f) Patient III.2 from the Australian cohort shows moderate photoreceptor dysfunction involving both the rod and cone systems. The DA 3.0, 30-Hz flicker, and LA 3.0 have b-wave amplitudes reduced to 50% of normal. The DA0.01 has blink artifact affecting interpretation. The pattern ERG (assessing macular function, not shown) was extinguished for the 15° field responses. The combination of electrophysiology results, OCT loss of ellipsoid zone, and reduced visual acuity led to classification as a cone–rod pattern of dysfunction. (h) Normal age-matched ISCEV standard ERG recordings are shown for comparison. These illustrate normal amplitudes and latencies for the scotopic (rod dependent system) DA 0.01, DA 3.0, and DA 12.0, and photopic (cone dependent system) 30-Hz flicker and LA 3.0. (i–k) OCT and fundus imaging for patient II.3, Australian cohort. (i) OCT shows thickening of the retina with loss of the normal retinal layer reflectance pattern and vitreous condensation (band) attaches to an epiretinal membrane. (j) The standard 50° fundal retinal image shows optic atrophy, marked retinal vascular attenuation, and pigmentary retinopathy. There is significant vitreous condensation overlying the retina obscuring some detail. (k) Ultrawide field image highlighting the extent of the pigmentary changes, which extend to the equator. (l) OCT retinal nerve fiber layer (RNFL), patient II.1 from the Delaware cohort, showing optic nerve edema and thickening of the peripapillary retinal nerve fiber layer (arrows). (m) Macula OCT, patient II.1 from the Delaware cohort, showing thickening of retina and reduction of the retinal lamination reflectance layers. (n) Goldmann visual fields (left and right), patient II.1 from the Delaware cohort, showing constriction of visual fields. OCT, Optical coherence tomography; ERG, Electroretinogram; ISCEV, International Society for Clinical Electrophysiology of Vision; DA, Dark adapted; LA, Light adapted; Hz, Hertz; ms, Millisecond; Div, division; TMP, Temporal; SUP, Superior; NAS, Nasal; INF, Inferior; RNFLT, Retinal nerve fiber layer thickness.

Fig. 3

ALPK1 expression in pooled normal human ocular tissues, mouse retina, and mouse sweat glands. (a) Quantitative real-time polymerase chain reaction (PCR) data, normalized to the geometric mean of HSP90AB1, PPIA, and PSMC4, shows increased expression of ALPK1 in the RPE and optic nerve tissue relative to macula retina tissue. Error bars represent the standard deviation of expression in technical replicates. (b–f) Alpk1 expression in the retinal pigmented epithelium and photoreceptor cells. (b) Adult mouse retinal section stained with anti-Alpk1 (green, MyBioSource, cat #MBS001969), and DAPI (blue) shows expression of Alpk1 in the RPE and connecting cilium region of the photoreceptor cells. (c–e) Magnification of the RPE and photoreceptor layers. Centrin (red) is a pancentrin antibody that marks the connecting cilium, basal body, and adjacent centriole of the photoreceptor. The merged image shows the localization of Alpk1 to the basal body region of the photoreceptor cilium and the region of the adjacent centriole. (f) Magnification of the connecting cilium region. (g–r) Expression of Alpk1 in mouse sweat glands: immunostaining of mouse paw sweat glands with (g) DAPI labeling the nuclei of cells in the skin of the mouse paw; (h) anti-Alpk1 antibody (green, Proteintech, cat #19107-1-AP); (i) anti-ɑ smooth muscle actin (α-SMA, red) as a myoepithelial cell marker. (j) Merged image of three channels indicates the high expression of Alpk1 in myoepithelial cells of sweat glands. (k) Stacked image of multiple layers of the sweat gland with 0.5-μm Z-level using confocal microscope. (l) Mouse sweat gland histology illustrating the morphology and location. (m–r) Magnified regions of the sweat glands encompassed by the box in (k) and (l). Scale bars represent 50 μm in (b) and (k), and 10 μm in (c–e) and (q). CC connecting cilium, GC ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, IS inner segment of photoreceptors, ONL outer nuclear layer, OPL outer plexiform layer, OS outer segment of photoreceptors, RPE retinal pigmented epithelium.

Fig. 4

ALPK1 localization to the centrosomes and base of the cilia in cells and the impact of ALPK1 (c.710C>T, [p.Thr237Met]) variant on primary cilia assembly. (a–c) Antibody staining of fixed ARPE19 cells with anti-ALPK1 (green, Proteintech, cat #19107-1-AP) and anti-α-tubulin (red) antibodies in a mitotic cell shows the localization of ALPK1 in spindle poles. (d–f) Further immunostaining of ARPE19 cells with anti-γ-tubulin (red) confirmed the centrosomal localization of ALPK1 during mitosis. (g–i) Localization of ALPK1 in ARPE19 cells in interphase shows a diffuse cytosolic pattern and enrichment of ALPK1 in centrosomes of the cells, which colocalize with γ-tubulin. (j–l) ARPE19 cells were serum starved for 24 hours and immunostained with anti-ALPK1 (green, Proteintech, cat #19107-1-AP) and anti-acetylated-α-tubulin (red) showing the ALPK1 localization at the base of primary cilium. Cell nuclei are stained with DAPI (blue). (m) Fibroblast cells were treated with serum-free medium for 24 hours and labeled with primary cilia markers anti-acetylated-α-tubulin (red), anti-IFT88 (green), and DAPI (blue). Representative images of unaffected cells and affected cells carrying ALPK1 (c.710C>T, [p.Thr237Met]) variant show decreased number of ciliated cells in fibroblasts from affected patients compared with control. (n) Quantification of the ciliated cells in one unaffected control and two affected patients. The analysis showed significant decrease in assembly of primary cilia in the affected patients carrying ALPK1 (c.710C>T, [p.Thr237Met]) variant. The graphs show the means ± s.e.m. from three independent experiments where 100 cells were scored for each sample in each experiment. *p<0.05. Scale bars represent 5 μm in (a, d, g, j) and 10 μm in (m, n).